Abstract The microtubule cytoskeleton supports cell-division, cellular morphology and intracellular cargo transport. While the centrosome is a major site of microtubule nucleation in dividing cells, many differentiated cells harbor acentrosomal microtubule arrays. Prominent examples include germline cells, plant epidermis, epithelia and neurons. To understand cellular differentiation, it is crucial to learn how acentrosomal array architecture is set up to achieve a specific pattern of polymer numbers, length and dynamics that would support specialized cellular functions, often throughout the life of an organism. My laboratory studies the patterning of acentrosomal microtubules and its effect on cargo transport in C. elegans. We developed imaging tools and algorithms that allow an unprecedented level of analysis of microtubule organization in vivo and are compatible with live-imaging of cargo transport. We conducted unbiased screens to uncover novel microtubule regulators and are using genetics, imaging, and biochemical methods to understand their mechanisms. In parallel, we are investigating the biological significance of microtubule array patterns by examining the effects of these regulators on long-range intracellular transport. This proposal details the establishment of our experimental system, design and implementation of the screen, and preliminary characterization of select regulators. It then outlines our main goals for the next five years: completing the screen and elucidating the mechanisms that establish acentrosomal array architecture. These studies will determine how steady-state array architecture emerges from the control of single polymer nucleation and dynamics and how it is adapted to the function of specialized cells. Microtubules support fundamental biological processes such as cell migration, polarization and cargo transport. Hence, our work will have a significant impact: It will identify novel regulators that arrange the building blocks of acentrosomal arrays and it will determine the mechanisms by which they pattern the cytoskeleton and regulate transport. The involvement of cytoskeletal defects in numerous disorders suggests that in the long-range, our studies will help to shed light on mechanisms of cellular dysfunction that occurs during disease.